Traditional methods for monitoring electrical activity in complex systems, such as the neurons in living brains, use electrodes and therefore preclude the acquisition of high resolution spatiotemporal maps of activity. Even for single cells, the use of patch clamp technology, although very sensitive and capable of recording single action potentials, is invasive and does not permit recordings from very thin processes such as axons or dendritic spines. This has prompted the development of voltage-sensitive dyes (VSDs) whose optical properties change in response to membrane potential. These membrane-specific molecular probes can then be imaged with high speed cameras or laser scanning microscopes and the time-courses at multiple points within a specimen can be analyzed. Since the initial work on the squid giant axon (L. B. Cohen, B. M. Salzberg, H. V. Davila, W. N. Ross, D. Landowne, A. S. Waggoner, C. H. Wang, “Changes in axon fluorescence during activity: molecular probes of membrane potential,” J. Membr. Biol. 19, 1-36 (1974); I. Tasaki, “Energy transduction in the nerve membrane and studies of excitation processes with extrinsic fluorescence probes,” Ann. N.Y. Acad. Sci. 227, 247-267 (1974)), a variety of VSDs have been developed to study trans-membrane potential (TMP) primarily by linear optical measurements such as absorbance or fluorescence. These dyes can be excited with visible light and provide an effective method to image cell membranes and their physiology.
Non-linear optical phenomena are observed when a high intensity laser interacts with an optical material and are characterized by a probability that is proportional to the incident light intensity raised to a power greater than one. The use of these phenomena to detect TMP changes has been demonstrated. R. Araya, J. Jiang, K. B. Eisenthal, and R. Yuste, “The spine neck filters membrane potentials,”PNAS. 103, 17961-17966 (2006); I. Ben-Oren, G. Peleg, A. Lewis, B. Minke, and L. M. Loew, “Infrared nonlinear optical measurements of membrane potential in photoreceptor cells,” Biophys. J. 71, 1616-1620 (1996); O. Bouevitch, A. Lewis, I. Pinevsky, J. P. Wuskell, and L. M. Loew, “Probing membrane potential with non-linear optics.” Biophys. J. 65, 672-679 (1993); P. J. Campagnola, M.-d. Wei, A. Lewis, and L. M. Loew, “High resolution optical imaging of live cells by second harmonic generation,” Biophys. J. 77, 3341-3349 (1999); D. A. Dombeck, L. Sacconi, M. Blanchard-Desce, and W. W. Webb. “Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy.” J. Neurophysiol. 94, 3628-3636 (2005); A. C. Millard, L. Jin, A. Lewis, and L. M. Loew, “Direct measurement of the voltage sensitivity of second-harmonic generation from a membrane dye in patch-clamped cells,” Opt. Lett. 28, 1221-1223 (2003); A. C. Millard, L. Jin, M.-d. Wei, J. P. Wuskell, A. Lewis, and L. M. Loew, “Sensitivity of second harmonic generation from styryl dyes to trans-membrane potential,” Biophys. J. 86, 1169-1176 (2004); A. C. Millard, L. Jin, J. P. Wuskell, D. M. Boudreau, A. Lewis, and L. M. Loew, “Wavelength- and Time-Dependence of Potentiometric Non-linear Optical Signals from Styryl Dyes,” J. Membr. Biol. 208, 103-111 (2005); M. Nuriya, J. Jiang, B. Nemet, K. B. Eisenthal, and R. Yuste, “Imaging membrane potential in dendritic spines,” PNAS 103, 786-790 (2006).
Second harmonic generation (SHG) and two-photon excitation fluorescence (2PF) are both non-linear optical processes taking place in proportion to the square of the incident light intensity. 2PF is the non-linear form of one-photon excitation fluorescence and operates on a similar principle. In 2PF, two photons excite a fluorophore into a state corresponding to twice their individual energies; the fluorophore then relaxes to the lowest energy electronic excited state before emitting a fluorescent photon. The emission spectrum for this non-linear process is the same as in one-photon excitation. In contrast, SHG occurs instantaneously, when two photons are converted into one of twice the energy. SHG does not involve an excited state and therefore conserves energy; the harmonic photon is emitted coherently. There are several other interrelated differences between the two methods. The first is based on the order of the term that generates each optical phenomenon. The polarization of an optical material in the presence of a high intensity electric field is a power series with coefficients associated with the material's higher order electric susceptibilities. 2PF comes from the imaginary portion of the third-order term, depending linearly on the concentration of chromophore. SHG comes from the second-order term, depending quadratically on the concentration of the SHG-active chromophores, or “harmonophores”. Also, SHG is confined to loci lacking a center of symmetry, as provided, for example, by a cell membrane leaflet. P. Yan, A. C. Millard, M. Wei, and L. M. Loew, “Unique Contrast Patterns from Resonance-Enhanced Chiral SHG of Cell Membranes,” J. Am. Chem. Soc. 128, 11030-11031 (2006). 2PF does not have this symmetry constraint.
The chromophore used to generate both non-linear phenomena can be synthesized in several forms. Previous work has shown that some aminonaphthylethenylpyridinium-based dyes (“ANEP-based dyes”; FIG. 1A-C) can exhibit a relative signal change of 43% per 100 mV for SHG signals. A. C. Millard, P. J. Campagnola, W. Mohler, A. Lewis, and L. M. Loew, “Second Harmonic Imaging Microscopy,” Meth. Enzymol. 361, 47-69 (2003). It was also shown that SHG and 2PF signal sensitivity are wavelength dependent and scale linearly with applied voltage change. A. C. Millard, L. Jin, J. P. Wuskell, D. M. Boudreau, A. Lewis, and L. M. Loew, “Wavelength- and Time-Dependence of Potentiometric Non-linear Optical Signals from Styryl Dyes,” J. Membr. Biol. 208, 103-111 (2005). However, the kinetics of some previous dyes were too slow to follow action potentials. The hydrophobic tail of the dye, which is characteristic of the chromophore, and the head can, however, be tailored to optimize the relative signal change and its speed. Indeed, FM4-64 (FIG. 1D), a dye that provides SHG responses to membrane potential fast enough to follow action potentials, has a similar styryl chromophore with ethyl groups attached to both the amino tail and the quaternary ammonium head. D. A. Dombeck, L. Sacconi, M. Blanchard-Desce, and W. W. Webb. “Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy.”J Neurophysiol. 94, 3628-3636 (2005); M. Nuriya, J. Jiang, B. Nemet, K. B. Eisenthal, and R. Yuste, “Imaging membrane potential in dendritic spines,” PNAS 103, 786-790 (2006). However, FM4-64 has a relatively small sensitivity to membrane potential of only 7.5-10% per 100 mV.
There remains a need for dyes with improved (faster) response to membrane potential for SHG and 2PF, as well as the ability to be excited by 1064 nanometer femtosecond pulses. There is also a need for dyes for use in studying the dynamics of action potentials in axons and dendrites and the compositions of lipid membranes.